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Full text of "Cause of European Foul Brood"

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r. S. DEPARTMENT OF AGRICULTURE, 
BUREAU OF BHTOMOLOGY— CIRCULAR No. 157. 

L. O. HOWARD. 1 nlomologwl .ndChirf of Bur«u. 



THE CAUSE OF EUROPEAN FOUL BROOD. 



G. 1- WHITE, M. I).. Ph. D. 



i 



VlvERKliENT PRINTING OTFICE : t»II 




BUREAU OF ENTOMOLOGY. 

L. o. Howakd, Entomologist and Chief <>f Hunan. 

('. L. Mablatt, Entomologist and Acting Chief in Absence of chief. 

li. s. Clifton, Executive Assistant. 

W. V. Tastet, Chief clerk. 

F. II. Chittenden, in charge of truck crop and stored product insect in vt litigations. 

A. D. Hopkins, in charge of forest insect investigations. 

\V. I>. Hunter, ;'// charge of southern field crog insect investigations. 

F. M. Webster, in charge of cereal anil forage insect investigations. 

A. L. Quaintance, in charge of deciduous fruit insect investigations. 

E. F. Phillips, in charge of bee culture. 

D. M. Rookks, in charge of preventing spread of moths, field work. 

Uoi.i.a V. CURRIE, in charge of editorial work. 

Mabel Colcord, in charge of library. 

Investigations in Bee Culture. 

B. F. Phillips, in eliargc. 

G. F. White, J. A. Nelson, experts. 

G. S. Demutii. A. II. McCbay, X. E. Mcindoo, apicultural assistants. 

Pearle II. Garrison, prcparator. 

H. A. Surface, I). B. Casteel, collaborators. 



Circular No. 157. toned M i 

United States Department of Agriculture, 

BUREAU OF ENTOMOLOGY. 
L. O. HOWARD. Entomologist and Chief of Bureau. 



TIIK CAUSE OF EUROPEAN FOUL ItKOOO. 

Bj G l' White, M. l>.. Ph. I».. 
pert in Bacteriology. 

HISTORIC \i.. 

The purpose of this preliminary paper is to discuss briefly the ex- 
citing cause of European foul brood. 

With diseased materia] furnished by Cheshire, Cheyne made a 
bacteriologica] study of foul brood. The latter isolated and described 
;i bacterium from the brood dead of the disease and identified it as 
/,' /7///.v alvei. Cheshire agreed that the identification was correct. 
A joint paper by these men appeared in l NS -">. and for about a decade 
and a half thereafter Bacittw alvei was generally considered to lx> 
the cause of foul brood. The disease which, it i- believed, Cheyne 
Btudied is the one that has received the designation European foul 
brood. 

This disease is also believed to lie the one which William R. Howard 
worked upon and named " black brood." In 1900 be described as 
it- cause a bacterium to which he gave the name BadUtU miffi. 

It i> probable that Burn in 1906 was studying the same disease in 
Sw itzerland when he referred to " sour brood." Iii the disease he dis- 
covered a bacterium to which he referred a- the u gunthi ri-fonns." 

Maa—eii in L907, working probably with the same disease, appar- 
ently encountered the u gwUheri— forms " reported by Burn and 
named the new species Streptocoa us n, 

-mMi i mm hi: WORK uv THE WRITER ON El BOFKAH i "l i. BROOD. 

In 1907 the writer observed in European foul brood the bacterium 
which bail been named Strepi apt*. It was observed at the 

same time that there was present also another microorganism quite 

1 



2 THE CAUSE OF EUROPEAN FOUL BROOD. 

similar in appearance, but clearly different from the one which Burri 
had observed and Maassen had named. All attempts, however, to 
cultivate this new species were unsuccessful. Until more was known 
about this organism it was referred to in 1908 as "Bacillus F." 

In 1!)07 the writer demonstrated that American foul brood, an 
infectious brood disease, could be produced by feeding to healthy 
colonies pure cultures of Bacillus larva. This fact emphasized the 
probability that if European foul brood is also caused by a bac- 
terium this disease, too. could be produced by feeding pure cultures 
of the bacterium causing it. 

To demonstrate this, it was desirable in the first place to determine 
whether or not the virus of European foul brood was present and 
active in the diseased brood. Healthy bees were fed sirup which 
contained a suspension of the diseased brood, and European foul 
brood was produced. This showed that the diseased brood did con- 
tain the virus and that the disease could be produced by feeding. 
This being done, pure cultures of Bacillus alvei isolated from the dis- 
eased material were substituted for the diseased brood in the inocu- 
lation experiment. Pure cultures of Streptococcus apis were isolated 
and used likewise. Then cultures of Bacillus alvei and cultures of 
Streptococcus apis were used simultaneously in making the inocula- 
tions. European foul brood was not produced in any of the experi- 
ments where pure cultures of either or both organisms were used. 
These facts were sufficient to eliminate tentatively Bacillus alvei and 
Streptococcus apis from the list of possible causes of European foul 
brood, and to justify a strong suspicion that the microorganism which 
was referred to as " Bacillus Y " bore a causal relation to the disease. 
It was necessary, however, to reckon with other factors before a 
more definite statement could be made. 

A continuation of the work on the cause of European foul brood 
has yielded some interesting results. These will be briefly con- 
sidered in this preliminary paper. 

It has been observed in the examination of diseased brood that 
Bacillus alvei is frequently either absent, or present only in small 
numbers, in many of the larvae which seem from gross appearance to 
be dead of European foul brood. Such samples have been received 
as a rule from localities in which apparently the disease had only 
recently appeared. Frequently, also. Streptococcus apis seemed to 
be absent, or present only in small numbers, in many of the larva? 
which from gross appearance gave strong evidence of European foul 
brood. These bacteriological findings further strengthened the 
theory that neither Bacillus alvei nor Streptococcus apis is the pri- 
mary exciting cause of European foul brood. Other inoculation 
experiments were performed, using pure cultures of these two species. 



Till CAU8I "i i.i K'Ti \n lori. BB ». 

The results were always negative, confirming further similar results 
that had been previously obtained. 

I.i ' i \ r WORK l'.\ I Ml WHITER. 

While these facts were in m measure satisfactory, as i « - 1 i « ' i i • ^r 
strongly in indicate certain conclusions, there was -till wanting 
that degree of conclusiveness which i- always desired. By exp< 
mental inoculation and by the studj of the brood sick or dead of the 
disease which was artificially produced, however, considerable in- 
formation of the character hoped for has been obtained. The 
detail- of the technique used in making the inoculation will nol be 
given in this brief report. 

I KFEBIM1 Mil. I mm I i \ i iiiw. 

Diseased material from various localities was used for these inocu- 
lations. Some colonic- were fed diseased brood that contained, as 
revealed by cultural examinations, large* quantities of Bacillus nl >■<'*; 
oilier colonic- were fed diseased material containing large numbers 
of Streptococcus apis as shown by culture-, and -till others were 
fed diseased materia] which was demonstrated to contain neither 
/; illus ill '■< i nor Streptococcus apis. It was found that at the first 
appearance of the disease in each class of experiments the symptoms 
manifested by the 3ick larva? wen- the same. Larvae showing thi 
early symptoms were studied bacteriologically. The examinations 
showed that whether or not the diseased material \'^\ to the bees 
contained Bacillus <rl>-,i or Streptococcus apis these species were 
in the early stages of the disease either absent, or present in -mall 
numbers only. It is quite evident that the disease was not pro- 
duced l>y species of bacteria which were absent at this early stage 
of the disease. 

Continuing the bacteriological study of the larva? in the early 
stages of the disease, some new species were found to he present. 
One bacterium especially is frequently encountered. This species 
i- a small, -lender rod. apparently nonmotile and non-pore bearing. 
It i- t" V known by the name Bacterium eurydice. It- description 
will appear in a later publication. Experimental colonic- have been 
fed pure culture- of this species, !>ut no disease has been produced. 
Tentatively, therefore, this species is not to be regarded as the cause 
of European foul brood. 

Two oiher species of bacteria mighl he mentioned here as being 
of interest in connection with the study of the brood diseases. The 
lir-t to be mentioned is a motile, spore-bearing, easily cultivatable 
rod. It is to receive the name Bacillus orphi "••>•. It also will he de- 
scribed later. This species is occasionally found in very large num.- 



4 TI-IK CAISi: OF EUROPEAN FOUL BROOD. 

bers in samples of European foul brood. Feeding it in pure cultures 
has so far given negative results. This organism, then, can also 
be eliminated tentatively from the list of possible causes of this 
disease. The other species, mentioned by Lambotte in 1902 as 
Bacillus iik -xi ntci icns vulgaris^ may be said to belong to a group of 
bacteria found quite widely distributed in the apiary. Its infre- 
quency in diseased brood and its occurrence in small numbers readily 
eliminates this species from the list of possible causes. Most of the 
bacteria that are met with in the study of European foul brood were 
therefore excluded tentatively from the list of possible causes of the 
di-ease. 

Idie possibility of an ultramicroscopic virus was also considered. 
Brood sick or dead of European foul brood were removed from the 
combs and crushed. An aqueous suspension of this diseased material 
was then made in boiled water and filtered with the Berkefeld filter. 
The filtrate remained clear when incubated at different temperatures 
and cultures made from it produced no growth. Separate filt rations 
have been made of diseased brood received from various localities, but 
in no instance where healthy colonies were fed filtrate obtained in 
this way was European foul brood produced. The results of the ex- 
periments therefore justify the tentative conclusion that there is no 
filterable virus in European foul brood capable of producing the 
disease. To this extent, then, has the possibility of an ultramicro- 
scopic virus been eliminated. 

Having thus tentatively eliminated all the microscopically visible 
organisms except Bacillus Y from the list of possible causes and like- 
wise eleminated the probability of an ultramicroscopic virus, the 
tentative conclusion was naturally reached that this remaining micro- 
organism probably plays an important role in the etiology of Euro- 
pean foul brood. Such a conclusion was all the more imperative 
since this organism had been encountered so frequently in the brood 
of this disease and since, moreover, there had been no other factor 
observed to which the exciting cause could be attributed. 

SYMPTOMS MANIFESTED BY l.ARWE SICK OF EUROPEAN FOUL BROOD. 

This conclusion led to a more extended study of this microorganism 
in the disease produced experimentally. The presence of disease 
can usually be detected in the experimental colony during the week 
that the feeding is begun. The first indication of it may be that 
only a portion of a larva is seen in a cell (fig. 1). the remaining por- 
tion having been removed by the bees. Aside from an observation 
of this kind the earliest indication one get- from the macroscopic 
(gioss) examination is that sick larva 1 are found among the uncapped 
brood. One should acquaint himself, therefore, with certain symp- 
toms or signs manifested by sick larva 1 during the course of the dis- 



I Hi , \r-i 01 li R0P1 \N FOUL BROOD. 



,..,.,. bj which ii- presence can be diagnosed while the larva? are stiU 
alive. Some of these will now be considered. 




I Larra aica of European fool Fv 2 Healthj larva of the age repre 

.1, partlj removed bj the I »nted In ngnrc I (Original I 

. < >rii.-i n:i 1 I 

The length of time that a developing bee is sicfc of European foul 
brood i- variable. It can be stated in a general way thai the three 




■to, s sick larva of the age rapreaented 
in Bgnrea 2 :m«i t (Original.) 

.lay- ju-t preceding the time when 
a larva would ordinarily be capped 
is the most favorable period for 
making a diagnosis from the gross 
examination alone. 

When healthy larva' of the age 
represented in figures -. 3, i. and 
5 are slightly magnified a peristal- 




sirk larra with re if of call 
removed (Original i 



6 THE CAUSE OF EUROPEAN FOUL BROOD. 

sislike motion of theii bodies is easily -ecu. hut larvae of this same 
age when sick frequently exhibit a marked peristalsislike motion of 
their bodies which can be easily seen with the unaided eye. Some- 





FlG. 5. — Sick Larva which is more trans- 
parent than a healthy larva of the 
same age. (Original.) 



Fh.. (j. — Healthy larva' with dorsal wall 
turned toward the observer showing 
the narrow transparent area along the 
median dorsal wall. (Original, i 



times the color of the larva? assists in the selection of those that are 
diseased. If. instead of the glistening white or bluish-white appear- 
ance of healthy larvae, one observes some that are more transparent 

(fig. 5), or that possess a very 
slight yellowish tint, frequently 
such larva> are diseased. In the 
absence of the exaggerated peri- 
stalsislike movement, however, 
other tests should be applied, as 
the color symptom is at times de- 
ceptive. 

Figures 6 and 7 represent older 
larvae than the preceding. These 
have turned themselves in the cell 
so as to present a dorsal portion 
to the observer. The narrow and 
quite transparent area frequently 
seen along the dorsal median line 
of a larva serves often a useful 

FIG. 7.— Sick larva of the age represented p Urp ose ill the diagnosis of ElirO- 
in figure G. (Original, i * l ° 

pean toul brood. In a healthy 
larva (fig. 6) a pollen-colored intestinal mass is frequently plainly 
visible through this transparent dorsal area. Microscopically thi> 
mass is easily demonstrated to be largely pollen. If. however, upon 




I ill CAU8E OF El ROP1 w FOUL BH< l D. 7 

inspection this intestinal mass appears white or yellowish white, the 
presence of European foul brood is almost certain. A modification 
of this simple inspection method may often be profitably used. This 
consists simply in turning the larva in the cell with a pair of forceps 
until the median dorsal line is exposed t<» the observer. 

More frequently still, it will be found advantageous to remove the 
larva From the ©all with the forceps. With a little care this can be 
done, leaving the larva intact. It' the larva is diseased and the dis- 
ease is sufficiently advanced, a uliiti~.li intestinal content can very 
often U' plainly observed. In response to the muscular action <»f the 
larva this mass is frequently seen to be moved t<> and fro. 

v posrnvi ii -i roa rm ihm \m i\ living ulrvm. 

There is a sign represented in figure s which, in the experience 
of the writer, has proven thus far to be a positive symptom of Euro- 
pean foul brood. When the aire and condition of the diseased larva 
are favorable and these frequently are— the sign can be quite 
easily and conveniently demonstrated in this way: Select a larva to 
be tested, approximately of the age represented by figures •_'. :'>. and I ; 
remove it from the cell and place it upon glass, preferably with a 
dark background; with a dissecting needle in each hand and with 
their points near together, pierce with both needles the wall of the 
larva near it- head, avoiding the intestine; separate now the points 

of the needle- SO BS to tear the body wall CTOSSwise and continue to 

separate the two portions of the larva. If the larva is diseased and 
one i> successful in applying the test, it will be found that the in- 
testinal content will be -tripped from and pulled out of the posterior 
and blind end i A. fig. 1<M of the canal, obtaining results as repre- 
sented in figure s . In case of living, healthy larva' the intestinal 
content can not be removed in this way. 

This mass thus removed from the intestine, if examined micro- 
scopically, will Ih 1 found, in general, to consist of a white or slightly 
yellowish-white mass along the longitudinal axis. This central mass 

surrounded by a substance which is more or less transparent and 
mucuslike in appearance. The appearance of this outer portion, 
however, will vary in detail, depending in a great measure upon the 
stage of the disease when the examination is made. 

The force which i- applied in pulling the ma— from the intestine 
frequently causes this enveloping substance to -t retch and the in- 
closed whitish substance to break into segments a- represented in a 
of figure 8. This is an earlier stage of the disease than that repre- 
sented in either h or c of the same figure. 
3S171 — Cir. 167—12 2 



THE CAUSE OF EUROPEAN FOUL BROOD. 




Pig. S. 



-The intestinal content removed from larvffi sick of European foul brood but n 
yet dead of the disease. (Original.) 






Till. ( M BE "I M R0P1 vn I "i L BBOOD. 9 

It' the disease ia more advanced than either stage represented in 
figure s when this test ia applied, a portion of the intestinal content 

may How out in the for f a sac, the wall of which is eery easily 

broken. \\" I »*• i » broken the content of this saclike structure will flo* 
out as a rather thin whitish or yellow ish w hite fluid containing small 
whitish granules thai vary in size. If the disease is far advanced 
and tlic larva probably dead, the enveloping substance of the ii 
tinal contenl is so easily broken that often only whitish or yellow ish- 
white fluid with its granular content flows from the ruptured wall of 
the Ian a. 

Figures 2 and 6 represent healthy larva', and at these ages the seg- 
ments of the body are strongly marked off. Living larva' at thi 
ages, if suffering from European foul brood, frequently -how these 
markings less distinctly as represented in figures 3, I. and 7. This 
sign, too, may asssist in the -elec- 
tion of larva- that are suspected 

of being diseased. 

TIIK V VI I E OF EARLY SYMPTOMS IN THI 
HI L6R08I8 <>i I I BOPBAfl FOUL BBOOD. 

These symptoms of European 
foul brood are some of the more 
important ones that are observed 
in sick larva' or in those only 
recently dead. They are espe- 
cially valuable in the study of 
the disease in the experimental r e bmoim piuton ,in a stained a 

colony. They have not been used preparation rroni sick larva; al stage rep- 
. • . resented In a - (Original.) 

by the apiarist tor making a 

diagnosis. The symptoms of European foul brood that have been 
looked for by the bee keeper for the most part are the evidences of 
disease which obtain a- a result of the death of the brood. The 
post-mortem symptoms a- manifested by the dead larvae themselves 
have been the mosl positive evidences used by the bee keeper in 
diagnosing the disease. I; is hoped, however, that when they are 
well learned, the symptoms of European foul brood observed in 
living larva' and in those very recently dead may prove of value in 
the apiary as well as in the experimental colony. 

Practically all the later symptoms of European foul brood have 
also been observed during the course of the disease in the experi- 
mental colony. This fact i- used as evidence that the disease which 
was produced in the experimental colony was the same a- that encoun- 
tered in the apiary. Since the diseased material for making the 
inoculation- ha- been received from various source- and the disease 
produced was apparently the same in every case, the conclusion that 




10 THE CAUSE OF EUROPEAN FOUL BROOD. 

there is: but one disease present in the condition which is being called 
European foul brood is, therefore, still further confirmed. 

MICROSCOPIC STUDIES OF DISEASED LARY.E. 

Returning now to the discussion of European foul brood in the 
earlier stages, it should be emphasized that by a macroscopic exami- 
nation alone it is not always possible to make a positive diagnosis 
of the presence or absence of disease in a larva. During the very 
earliest period of infection it is impossible from the gross examina- 
tion alone to make a positive diagnosis of the presence of disease. 
Such is to be expected. About the time the larva dies there is a 
period at which one can not always be sure that the disease is present 
from a macroscopic examination alone. Between these stages there 
is a period in the course of the disease in the larva? during which it 
is usually possible to make a diagnosis positive from the gross exami- 
nation. Since a macroscopic examination alone is not always suffi- 
cient for making a positive diagnosis, one looks naturally to a micro- 
scopic examination for assistance. 

During the course of the disease in the experimental colony the 
microscopic picture presented in the examination of diseased larvae 
changes markedly. To begin the microscopic study, it is well to 
obtain the intestinal content as represented in a. figure 8. If a thin 
smear is made of the white growth-mass of this content and stained, 
it is found to consist almost entirely of forms represented in fig- 
ure 9. 

This organism is the one that the writer referred to in an earlier 
paper as " Bacillus F." All attempts to cultivate this new species 
on artificial media have thus far l>een unsuccessful. Since consider- 
able information has now been obtained concerning this organism 
the specific name " pin ton " is now substituted for the " F " in the 
term "Bacillus F" and the species will now be known as Bacillus 
pluton. This organism is an unusual one and the classification has 
not yet been definitely determined. The generic term " Bacillus,"' 
therefore, may. and probably will, be changed later. 

At the stage of the disease represented in a, figure 8, the majority 
of the individuals of this new species in general appear in stained 
preparations to be pointed at the ends (fig. 9). Some show both 
ends rather sharply pointed, others show only one end so pointed, 
the other end being rounded, while still others show both ends 
rounded. The individuals having this general form vary much in 
size. They are as a rule 1 \i or less in length, the breadth being 
about one-half the length. Forms in pairs frequently occur in a 
smear preparation made at this stage of the disease. These paired 
forms vary markedly in size and shape. (See fig. 9.) Accompany- 



I II I CAUS1 "I M ROlM \N I «>i i. UR(K ID. 



11 



bug />>" illus phit. . /: terium enri P frequently found at this 

stage of the disease, but in comparatively small numbers. 

If the intestinal content in a later stage of the disease for exam- 
ple, that represented in 6, lii, r| ip' B is examined microscopically, 
Bacillus pin ton is still found in verj targe numbers, and Bacterium 
eurydicx when present will be relatively veri much increased in num- 
bers. V similar examination of the intestinal content represented in 
r. figure s . will usually show Bacillus pint on in large numbers, Bac- 
terium eurydin in increased numbers, and in addition one may find 
Hug alri'i in comparatively small numbers. 




I 10 in.it i.- drawing representing o long n • <( n larra at nn enrlr 

■ >f infection. The position of the li »m, BaviUut pluton, la aloi 

near the perltropblc membrane. (Original 

By examining the fluid mass which flows from the body of a 
Larva when the disease is far advanced and Wie body wall is broken, 
pne usually find-, together with Bacillus pluton, bacteria of different 
species in considerable numbers. 

From this point on in the decay of the larva' the relative propor- 
tion of the different microorganisms present varies markedly. When 
Bacillus nl'-ii is present it increases verj rapidly in proportion to 
the other-. This rapid increase of Bacillus alvei in the larvae after 
the death of the larva; account- in a large measure for the frequency 
with which this species is mentioned in report.- on this disease. 



12 THE CAUSE OF EUBOPEAN FOUL BROOD. 

Id living larv;v. therefore, in which European foul brood can be 
diagnosed from gross examination, it is found that bacteria usually 
accompany BacUlus phiton. This fact made desirable the study of 
the diseased larvae in still earlier stages of the infection, i. e., during 

the period of incubation. This was done culturally in part, but prin- 
cipally by fixing and sectioning the younger larvae from strongly 
infected experimental colonies. From such sections it was observed 
that BacUlus pluton was the first invader of the healthy larvae. 

Figure 10 represents schematically the condition in the larva' at 
an early stage of infection. Tn this figure fg represents the foregut; 
iikj the midgut, and Kg the hindgut. At this age of the larvae the 
posterior end of the midgut is closed, as represented at b. Tn the 
same figure, m represents that portion of the intestinal content lying 
in contact with the wall of the intestine: /. the central portion of the 
drawing, represents the food taken at this age; and p represents 
what seems to be a peritrophic membrane between the enveloping 
substance, m, and the paplike food substance. /. of the midgut. 

In the growth of Bacillus phiton this parasite very early takes a 
position along the. peritrophic membrane p. and just central to it 
(fig. 10). At this early stage of its growth this microorganism pre- 
sents in general an appearance of being rod shaped with a strong 
tendency to <rrow in chains. As the disease advances and the growth- 
mass of this organism increases, the central portion of the lumen of 
the intestine becomes filled by a solid growth which is made up very 
largely of Bacillus pluton. During this stage of the disease the con- 
tent can be removed from the posterior blind end of the midgut, as 
shown in figure 8. The relation of the central growth-mass to the 
surrounding mucuslike-appearing mass represented in a, b, and c 
of figure 8 is well demonstrated microscopically by sectioning these 
intestinal masses. 

From the studies made thus far it would seem that Bacillus phiton 
is easily killed by heat. 

PROBABLE EXPLANATIONS OF ERRORS AS TO THE EXCITING CAUSE OF 

EUROPEAN FOUL BROOD. 

It is quite probable that others at different times have observed this 
new species. I!<n illns pluton, but have failed to differentiate it from 
bacteria which were present and which appeared in the cultures 
made, leading them thus to erroneous statements concerning the dis- 
ease and its exciting cause. For example. William R. Howard may 
have seen this organism microscopically in his so-called " black 
brood." but failed to differentiate it from some bacterium — BacUlus 
milii or Bacillus alvei — which he cultivated on artificial media. 
Burri may have seen it in the so-called "sour brood " and mistaken it 



1 III CAU81 01 m BOP] w FOUL BBOOD. 1 .''> 

for the u ffuntheri- forms* 1 which he observed in hia cultures. Maa Ben 
mentions some difficulty experienced at times in obtaining Strep 
coccus apis from brood which on microscopic examination seemed to 
contain this bacterium. To explain this difficulty, he advanced the 
supposition that the Streptococcus was probabh killed by acid pro- 
duced by itself. The difficulty probably could be as "''II explained 
by supposing thai Maassen failed to differentiate this parasite from 
the bacterium which he cultivated and described as Streptocod us apis. 

is Mil i:i Mci;i iiiw ONI DISJ \-i in nil CONDITION KNOWN \- 
l l Ri >PE w FOUL BBOOD i 

The question now arises whether or nol there is more than one 
disease in the condition now known a- European foul brood. In 
Switzerland and in Germany there has been a tendency to diagnose 
the diseased brood in which Bacillus alvei is found as the foul brood 
of Cheshire and Cheyne and the diseased brood in which Strepto- 
apis is found as "sour brood." From the facts at hand the 
writer is strongly inclined to believe that these two conditions are 
only the one disease, known in America a- European foul brood. 
Enough evidence has not yet been obtained, however, to -peak with 
complete positiveness on this point. 

\- secondary invaders some of the specie- of bacteria mentioned 
in this paper may and probably do exert an influence on the course 
of the disease in the larva and in the colony. To what extent these 
bacteria modify the disease is yet to lie determined. Should it he 
found that Bacillus alvei actually causes an infection- brood disease, 
then such a disease should 1h> called European foul brood, and the 
disease caused by Bacillus pluton would have to Ik- differentiated 
from it. 

Further detail- will not he given in thi> preliminary announce- 
ment hut will he included in more technical paper- which are being 

prepared. 

-t M m un \M> CON< ii BI0N8. 

The -tep- taken in the writer'- endeavor to find the cause of Kuro- 
pean foul brood may l>c briefly summarized a- follow-: 

lit Hit, it his alvei) which has been so generally spoken of as the 
cause of foul brood, was isolated from diseased brood, and pure cul- 
ture- of the organism in both the vegetative and -pore forms were 
repeatedly \\-A to colonies of healthy U-e- with the result that foul 
brood was not produced in any instance. This fact cast a suspicion 
that Bacillus alvei was probably not the cause of a disease. 

(2) By a study of many larva- in -ample- of European foul brood 
it was frequently found that there were larvae apparently dead of the 
disease that contained Bacillus alvei only in small numbers or not at 



14 THE CAUSE OF EUROPEAN FOUL BROOD. 

all. This increased the suspicion that Bacillus <>/>■<; was not the 
exciting cause of the disorder. 

(3) In 1907 the writer proved that by feeding pure cultures of 
Bacillus larval to healthy bees American foul brood could be produced. 
This fact still further emphasized the doubt that was already enter- 
tained concerning the possibilities of Bacillus alvei in the etiology of 
European foul brood. 

(4) By feeding diseased larva' to healthy colonies it was found that 
European foul brood could be artificially produced, showing that this 
disease, too, could be produced by feeding, and that the virus was 
contained in the diseased brood. 

( ."> ) The sick larvae of the disease thin artificially produced were 
frequently found, when examined, to be free from Bacillus at 
This evidence, too, was damaging to the theory that Bacillus alvei is 
the cause of a brood disease. 

(6) Bacillus aire/ in this way was tentatively eliminated from the 
list of possible exciting causes of European foul brood. In a quite 
similar manner the other bacteria — Streptococcus apis. Bacillus me- 
sentericus vulgaris, Bacillus orpkeus, and Bacteiiuni earn/dice — were 
likewise eliminated from the list. 

(7) Considerable quantities of filtrate from aqueous suspensions of 
crushed diseased larvae were fed to healthy colonies and in no instance 
was European foul brood produced. This eliminated tentatively the 
probability of there being an ultramicroscopic virus in European foul 
brood capable of producing the disease. 

(8) Bacillus pluton, therefore, was the only factor that was not 
so eliminated from the list of possible exciting causes of the disease 
and became thus the probable exciting cause of European foul brood 

(9) "When this organism was studied in larva 1 in which the disease 
could be suspected by inspection alone, one or more species of bacteria 
were sometimes found to be present als"o. These, when present, how- 
ever, occurred in relatively small number-. 

(10) The disease was then studied in a still earlier stage; i. e.. 
before its presence could be detected by gross examination of the 
larva 1 . This was done by cultures in part, but principally by fixing 
and sectioning larva during the incubation period of the disease. 
This study demonstrated that in the production of the disease Bacillus 
pluton was the first invader of the healthy larvae. 

It will be noticed, therefore, that in the determination of the pri- 
mary exciting cause of European foul brood two objects were accom- 
plished : (1) All the factors in the list of possible exciting causes of 
the disease were eliminated except the one organism Bacillus pluton, 
and (2) by the study of infected larva 1 soon after the infection took 
place, this parasite was found to be the first invader. 



I II v CAUSI i»i 1 l'KOl'1 W FOUL BROOD. 



i:» 



As b conclusion, it i- the belief of the writer thai sufficient evidence 
has noM been obtained to justify the statement thai Bacillus pluton 
is the primary exciting cause oi a brood disease. Thia brood dise 
is now generall) known in America as European foul brood. Hi; 
opinion is rendered in accordance with view- no* generally accepted 
relative to the etiology of animal disea 

There are, then, three principal brood di • Two of thi 

American foul brood, caused l»\ Bacillus larva 1 , and European foul 
brood, caused by Bacillus pluton are known l<> be infectious. Prom 
these two diseases there must be differentiated the third one, an 
apparently noninfectious disorder, the so-called w pickled brood." 
Larva? dead of this latter disease are practically free from micro 
organisms. The exciting cause of thia disorder is not yet known. 

Approved : 

.1 \mi a Wilson, 

s ■ , ij of . I '/' U ult in . , 

Washington, D. ('.. March 28, 1912. 



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